Assignment of biochemical functions to glycosyl transferase genes which are essential for biosynthesis of exopolysaccharides in Sphingomonas strain S88 and Rhizobium leguminosarum.
Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The spsK gene of Sphingomonas strain S88 and the pssDE genes of Rhizobium leguminosarum were identified as encoding glucuronosyl-(B1-->4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the spsK gene and the pssDE genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that spsL codes for a glucosyl-(beta1-->4)-glucuronosyl transferase in Sphingomonas and that pssC codes for a glucuronosyl-(beta1-->4)-glucuronosyl transferase in R. leguminosarum. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C55-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.
Pollock
T J
TJ
Shin-Etsu Bio, Inc., San Diego, California 92121, USA. 76554.3460@compuserve.com
van Workum
W A
WA
Thorne
L
L
Mikolajczak
M J
MJ
Yamazaki
M
M
Kijne
J W
JW
Armentrout
R W
RW
D002240:Carbohydrate Sequence; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D016695:Glycosyltransferases / Q000235:genetics*; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000378:metabolism; D016961:Rhizobium leguminosarum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D011135:Polysaccharides, Bacterial; D016695:Glycosyltransferases